Abstract Connexin (Cx) proteins form hexameric hemichannels (HCs) that dock end-to-end to form gap junction channels (GJCs) across the extracellular gap, allowing intercellular exchange of nutrients, metabolites, ions and signaling molecules. Over the last 3 decades our research program has explored the structure and regulation of two Cx isoforms, Cx43 and Cx26. Each is found in many tissues. The former most notably mediates electrical conduction between cardiac myocytes, enabling the normal heartbeat, but also mediating potentially fatal cardiac arrhythmias. The latter, is most well known for its role in the inner ear; mutations of Cx26 are the predominant cause of inherited deafness. Over the last dozen years we have focused on the regulation of Cx26 channels during tissue injury, associated with Ca2+ overload and acidic pH. We determined X-ray structures of the human Cx26 GJC with and without bound Ca2+. To our surprise, the two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Computational analysis revealed that the binding of Ca2+ ions creates a positive electrostatic barrier that blocks K+ permeation. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion. To examine pH-mediated gating of Cx26 GJCs we used cryoEM and single-particle image analysis coupled with H/D exchange and crosslinking mass spectrometry. The results support a steric ?ball-and-chain? mechanism in which association of the acetylated N-termini form a pore-occluding, gating particle. Building on this rigorous structural and biophysical analysis of WT channels, we are now in a position to explore (1) the effects of deafness-causing mutations of Cx26 that involve residues that participate in Ca2+ coordination, (2) the effects of mutations of residues implicated in pH regulation, (3) the structure of undocked hemichannels and (4) structures of other connexins, particularly Cx32, mutations of which cause peripheral neuropathy, and also the cardiac connexins Cx43, in the working myocardium, and Cx40, in the specialized conducting tissue. Our structural studies utilize X-ray crystallography, cryoEM, crosslinking, H/D exchange mass spectrometry (HDX) and EPR spectroscopy in a synergistic manner. Functional studies include electrophysiology and proteoliposome-based transport assays. Our research program is fortified by fruitful collaborations with 3 experts: Drs. Andrew Harris (electrophysiology and functional assays), Patrick Griffin (HDX mass spectrometry) and David Cafiso (EPR spectroscopy). Our proposed research provides an opportunity to understand aspects of GJC and HC channel function that have been long-desired, and to initiate exploration of how those structure-function properties operate in several members of the Cx family. Given the importance of proper Cx channel function in development, pathophysiology and response to disease and trauma, this understanding will have substantial biomedical impact. !